Implanted scaffold-free prevascularized constructs promote tissue repair.

To evaluate the anastomotic potential of prevascular tissue constructs generated from scaffold-free self-assembly of human endothelial and fibroblast cells, tissue constructs were implanted into athymic mice and immune-competent rats. Analysis of xenografts placed into hind limb muscle defects showed vascular anastomotic activity by 3 days after implantation and persisting for 2 weeks. Integration of the implanted prevascular tissue constructs with the host circulatory system was evident from presence of red blood cells in the implant as early as 3 days after implantation. Additionally, analysis of 3-day xenografts in the rat model showed activation of skeletal muscle satellite cells based on Pax-7 and MyoD expressions. We conclude that prevascular tissue constructs generated from scaffold-free self-assembly of human endothelial and fibroblast cells are a promising tool to provide both vascular supply and satellite cell activation toward the resolution of skeletal muscle injury.

Self-assembly of prevascular tissues from endothelial and fibroblast cells under scaffold-free, nonadherent conditions.

To advance the emerging field of bioengineered prevascularized tissues, we investigated factors that control primary vascular network formation in scaffold-free, high-density cell suspension-derived tissues. Fabricating primary vascular networks in a scaffold-free system requires endothelial cells (ECs) and extracellular matrix (ECM)-producing cells that act together to elaborate a permissive matrix. We report findings on the effects to vascular patterning induced by altering the ratio of human endothelial to human fibroblast cells. Analysis revealed that a 1:4 ratio of ECs to fibroblasts resulted in the synthesis of an ECM permissive for organization of primary vascular networks that recapitulated the pattern of primary vascular networks observed in vivo. Importantly this work highlighted the significance of tension in the organization of vascular networks in prevascularized tissues. To our knowledge our in vitro studies are the first to demonstrate the formation of two distinct vascular patterns in an initially homogenous culture system. Specifically, we demonstrate that within our constructs, vascular networks formed with distinct directional orientations that reflect self-assembly-mediated tension. Further, our studies demonstrate that treatment of prevascularized tissues with matrix-promoting factors such as transforming growth factor beta 1 (TGFß1) increases tissue strength without altering vascular network patterning. Together, the ability to generate prevascularized tissues from human cells in scaffold-free systems and the ability to enhance the strength of the constructs with matrix-promoting factors represent advances to the potential translational utility of prevascularized tissues both as subcutaneous implants and in surgical scenarios requiring the application of tension to the tissue construct.

3D printing facilitated scaffold-free tissue unit fabrication.

Tissue spheroids hold great potential in tissue engineering as building blocks to assemble into functional tissues. To date, agarose molds have been extensively used to facilitate fusion process of tissue spheroids. As a molding material, agarose typically requires low temperature plates for gelation and/or heated dispenser units. Here, we proposed and developed an alginate-based, direct 3D mold-printing technology: 3D printing microdroplets of alginate solution into biocompatible, bio-inert alginate hydrogel molds for the fabrication of scaffold-free tissue engineering constructs. Specifically, we developed a 3D printing technology to deposit microdroplets of alginate solution on calcium containing substrates in a layer-by-layer fashion to prepare ring-shaped 3D hydrogel molds. Tissue spheroids composed of 50% endothelial cells and 50% smooth muscle cells were robotically placed into the 3D printed alginate molds using a 3D printer, and were found to rapidly fuse into toroid-shaped tissue units. Histological and immunofluorescence analysis indicated that the cells secreted collagen type I playing a critical role in promoting cell-cell adhesion, tissue formation and maturation.

Scaffold-free tissue engineering: organization of the tissue cytoskeleton and its effects on tissue shape.

Work described herein characterizes tissues formed using scaffold-free, non-adherent systems and investigates their utility in modular approaches to tissue engineering. Immunofluorescence analysis revealed that all tissues formed using scaffold-free, non-adherent systems organize tissue cortical cytoskeletons that appear to be under tension. Tension in these tissues was also evident when modules (spheroids) were used to generate larger tissues. Real-time analysis of spheroid fusion in unconstrained systems illustrated modular motion that is compatible with alterations in tensions, due to the process of disassembly/reassembly of the cortical cytoskeletons required for module fusion. Additionally, tissues generated from modules placed within constrained linear molds, which restrict modular motion, deformed upon release from molds. That tissue deformation is due in full or in part to imbalanced cortical actin cytoskeleton tensions resulting from the constraints imposed by mold systems is suggested from our finding that treatment of forming tissues with Y-27632, a selective inhibitor of ROCK phosphorylation, reduced tissue deformation. Our studies suggest that the deformation of scaffold-free tissues due to tensions mediated via the tissue cortical cytoskeleton represents a major and underappreciated challenge to modular tissue engineering.

VEGF-mediated phosphorylation of eNOS regulates angioblast and embryonic endothelial cell proliferation.

To evaluate potential roles of nitric oxide (NO) in the regulation of the endothelial lineage and neovascular processes (vasculogenesis and angiogenesis) we evaluated endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) expression in 7.2-8.5 days post-coitum (dpc) mouse embryos. Analysis revealed that p-eNOS((S1177)) but not P-eNOS((S617)) or P-eNOS((T495)) was expressed in a subpopulation of angioblasts (TAL-1(+)/Flk-1(+)/CD31(-)/CD34(-)/VE-Cadherin(-)) at 7.2 dpc. A role of the VEGF/Akt1/eNOS signaling pathway in the regulation of the endothelial cell (EC) lineage was suggested by the strong correlation observed between cell division and p-eNOS((S1177)) expression in both angioblasts and embryonic endothelial cells (EECs, TAL-1(+)/Flk-1(+)/CD31(+)/CD34(+)/VE-Cadherin(+)). Our studies using Akt1 null mouse embryos show a reduction in p-eNOS((S1177)) expression in angioblast and EECs that is correlated with a decrease in endothelial cell proliferation and results in changes in VEGF-induced vascular patterning. Further, we show that VEGF-mediated cell proliferation in Flk-1(+) cells in allantoic cultures is decreased by pharmacological inhibitors of the VEGF/Akt1/eNOS signaling pathways. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation on Ser1177 regulates angioblast and EEC division, which underlies the formation of blood vessels and vascular networks.

Recruitment of bone marrow-derived valve interstitial cells is a normal homeostatic process.

Advances in understanding of the maintenance of the cardiac valves during normal cardiac function and response to injury have led to several novel findings, including that there is contribution of extra-cardiac cells to the major cellular population of the valve: the valve interstitial cell (VIC). While suggested to occur in human heart studies, we have been able to experimentally demonstrate, using a mouse model, that cells of bone marrow hematopoietic stem cell origin engraft into the valves and synthesize collagen type I. Based on these initial findings, we sought to further characterize this cell population in terms of its similarity to VICs and begin to elucidate its contribution to valve homeostasis. To accomplish this, chimeric mice whose bone marrow was repopulated with enhanced green fluorescent protein (EGFP) expressing total nucleated bone marrow cells were used to establish a profile of EGFP(+) valve cells in terms of their expression of hematopoietic antigens, progenitor markers, fibroblast- and myofibroblast-related molecules, as well as their distribution within the valves. Using this profile, we show that normal (non-irradiated, non-transplanted) mice have BM-derived cell populations that exhibit identical morphology and phenotype to those observed in transplanted mice. Collectively, our findings establish that the engraftment of bone marrow-derived cells occurs as part of normal valve homeostasis. Further, our efforts demonstrate that the use of myeloablative irradiation, which is commonly employed in studies involving bone marrow transplantation, does not elicit changes in the bone marrow-derived VIC phenotype in recipient mice.

Fusion of uniluminal vascular spheroids: a model for assembly of blood vessels.

We evaluated the self-assembly properties of uniluminal vascular spheroids having outer layers of vascular smooth muscle cells and a contiguous inner layer of endothelial cells lining a central lumen. We showed that while pairs of uniluminal vascular spheroids suspended in culture medium fused to form a larger diameter spheroidal structure, spheroids in collagen hydrogels formed elongated structures. These findings highlight the potential use of uniluminal vascular spheroids as modules to engineer blood vessels. We also demonstrate that uniluminal vascular spheroid fusion conforms to models describing the coalescence of liquid drops. Furthermore, the fusion of uniluminal vascular spheroids in vitro closely resembled the in vivo process by which the descending aorta forms from the fusion of the paired dorsal aortae during embryonic development. Together, the findings indicate that tissue liquidity underlies uniluminal vascular spheroid fusion and that in vivo anastomosis of blood vessels may involve a similar mechanism.

Organ printing: tissue spheroids as building blocks.

Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.

Resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function.

The purpose of the workshop was to identify still obscure or novel cellular components of the lung, to determine cell function in lung development and in health that impacts on disease, and to decide promising avenues for future research to extract and phenotype these cells. Since robust technologies are now available to identify, sort, purify, culture, and phenotype cells, progress is now within sight to unravel the origins and functional capabilities of lung cells in developmental stages and in disease. The Workshop's agenda was to first discuss the lung's embryologic development, including progenitor and stem cells, and then assess the functional and structural cells in three main compartments of the lung: (1) airway cells in bronchial and bronchiolar epithelium and bronchial glands (basal, secretory, ciliated, Clara, and neuroendocrine cells); (2) alveolar unit cells (Type 1 cells, Type 2 cells, and fibroblasts in the interstitium); and (3) pulmonary vascular cells (endothelial cells from different vascular structures, smooth muscle cells, and adventitial fibroblasts). The main recommendations were to: (1) characterize with better cell markers, both surface and nonsurface, the various cells within the lung, including progenitor cells and stem cells; (2) obtain more knowledge about gene expression in specific cell types in health and disease, which will provide insights into biological and pathologic processes; (3) develop more methodologies for cell culture, isolation, sorting, co-culture, and immortalization; and (4) promote tissue banks to facilitate the procurement of tissue from normal and from diseased lung for analysis at all levels.

VEGF-mediated fusion in the generation of uniluminal vascular spheroids.

Embryonic mouse allantoic tissue (E8.5) was cultured in hanging drops to generate a three-dimensional vascular micro-tissue. The resulting tissue spheroids had an inner network of small diameter vessels expressing platelet endothelial cell adhesion molecule-1 (PECAM-1) and an outer layer of cells expressing SMalphaA, SM22-alpha, and SM-MHC. In a subsequent phase of culture, the fusion-promoting activity of vascular endothelial growth factor (VEGF) was used to transform the inner network of small diameter endothelial tubes into a contiguous layer of cells expressing PECAM-1, CD34, and VE-cadherin that circumscribed a central lumen-like cavity. The blood vessel-like character of the VEGF-treated spheroids was further demonstrated by their physiologically relevant vasodilatory and contractile responses, including contraction induced by KCl and relaxation stimulated by high-density lipoproteins and acetylcholine-induced nitric oxide production.

Fibulin-1 is required for morphogenesis of neural crest-derived structures.

Here we report that mouse embryos homozygous for a gene trap insertion in the fibulin-1 (Fbln1) gene are deficient in Fbln1 and exhibit cardiac ventricular wall thinning and ventricular septal defects with double outlet right ventricle or overriding aorta. Fbln1 nulls also display anomalies of aortic arch arteries, hypoplasia of the thymus and thyroid, underdeveloped skull bones, malformations of cranial nerves and hemorrhagic blood vessels in the head and neck. The spectrum of malformations is consistent with Fbln1 influencing neural crest cell (NCC)-dependent development of these tissues. This is supported by evidence that Fbln1 expression is associated with streams of cranial NCCs migrating adjacent to rhombomeres 2-7 and that Fbln1-deficient embryos display patterning anomalies of NCCs forming cranial nerves IX and X, which derive from rhombomeres 6 and 7. Additionally, Fbln1-deficient embryos show increased apoptosis in areas populated by NCCs derived from rhombomeres 4, 6 and 7. Based on these findings, it is concluded that Fbln1 is required for the directed migration and survival of cranial NCCs contributing to the development of pharyngeal glands, craniofacial skeleton, cranial nerves, aortic arch arteries, cardiac outflow tract and cephalic blood vessels.

Transplanted human cord blood cells generate amylase-producing pancreatic acinar cells in engrafted mice.

OBJECTIVES: Pancreatic acinar cells and hepatocytes arise from the same cell population located within the embryonic endoderm. It has been reported that a multipotent population of liver cells is capable of differentiating into pancreatic cells. Recent studies revealed that murine and human hematopoietic cells could generate hepatocytes in vivo. Based on this developmental proximity between liver and pancreatic acinar cells, we examined whether human cord blood (CB) cells can generate pancreatic cells in vivo using a murine xenograft model. METHODS: We transplanted 1 x 10 CD34 human CB cells into "conditioned" newborn nonobese diabetic-severe combined immunodeficiency/beta-2 microglobulin-null mice via facial vein injection and, 3 to 4 months later, examined the pancreata from recipient mice showing high-level human multilineage hematopoietic engraftment in the bone marrow. RESULTS: Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses revealed human amylase mRNA and protein expression, respectively, in the pancreata from recipient mice. Using fluorescence in situ hybridization, we identified human alpha-satellite, DNA-positive cells with a morphology characteristic of pancreatic acinar cells. We also identified cells in paraffin sections of the pancreata that expressed amylase mRNA, had morphological characteristics of acinar cells, and contained human but not mouse centromeric DNA. CONCLUSION: These findings establish that human umbilical CB cells are capable of generating pancreatic acinar cells via a nonfusion mechanism.

The genetics of vasculogenesis.

To identify genes important to the process of vasculogenesis, we have used a novel meta-analysis approach to evaluate retrospectively the embryonic vascular anomalies observed in over 100 mouse gene knockout studies. Through application of this method, termed Approach for Ranking of Embryonic Vascular Anomalies (AREVA), 12 genes were determined to be critical to vasculogenesis. Importantly, when the 12 genes were considered with respect to VEGF-VEGFR signalling, an integrated network centreing on the ShcA/Ras/Raf/Mek/Erk pathway became apparent. Herein, we discuss how the 12 vasculogenesis-critical genes influence specific stages in the process of vasculogenesis.

Hematopoietic origin of fibroblasts/myofibroblasts: Its pathophysiologic implications.

Tissue fibroblasts/myofibroblasts play a key role in growth factor secretion, matrix deposition, and matrix degradation, and therefore are important in many pathologic processes. Regarding the origin of tissue fibroblasts/myofibroblasts, a number of recent in vivo transplantation studies have suggested the bone marrow as the source of fibroblasts/myofibroblasts in liver, intestine, skin, and lung. Because bone marrow cells are thought to contain 2 types of stem cells (ie, hematopoietic stem cells [HSCs] and mesenchymal stem cells), it is important to determine which type of stem cells is the source of fibroblasts/myofibroblasts. To address this issue, we have carried out a series of studies of tissue reconstitution by single HSCs. By transplanting clones derived from single HSCs expressing transgenic enhanced green fluorescent protein, we found that fibroblasts/myofibroblasts in many organs and tissues are derived from HSCs. This brief note summarizes these findings and discusses clinical and experimental perspectives generated by this newly identified differentiation pathway of HSCs.

Targeted disruption of cubilin reveals essential developmental roles in the structure and function of endoderm and in somite formation.

BACKGROUND: Cubilin is a peripheral membrane protein that interacts with the integral membrane proteins megalin and amnionless to mediate ligand endocytosis by absorptive epithelia such as the extraembryonic visceral endoderm (VE). RESULTS: Here we report the effects of the genetic deletion of cubilin on mouse embryonic development. Cubilin gene deletion is homozygous embryonic lethal with death occurring between 7.5-13.5 days post coitum (dpc). Cubilin-deficient embryos display developmental retardation and do not advance morphologically beyond the gross appearance of wild-type 8-8.5 dpc embryos. While mesodermal structures such as the allantois and the heart are formed in cubilin mutants, other mesoderm-derived tissues are anomalous or absent. Yolk sac blood islands are formed in cubilin mutants but are unusually large, and the yolk sac blood vessels fail to undergo remodeling. Furthermore, somite formation does not occur in cubilin mutants. Morphological abnormalities of endoderm occur in cubilin mutants and include a stratified epithelium in place of the normally simple columnar VE epithelium and a stratified cuboidal epithelium in place of the normally simple squamous epithelium of the definitive endoderm. Cubilin-deficient VE is also functionally defective, unable to mediate uptake of maternally derived high-density lipoprotein (HDL). CONCLUSION: In summary, cubilin is required for embryonic development and is essential for the formation of somites, definitive endoderm and VE and for the absorptive function of VE including the process of maternal-embryo transport of HDL.

An in vivo analysis of hematopoietic stem cell potential: hematopoietic origin of cardiac valve interstitial cells.

Recent studies evaluating hematopoietic stem cell (HSC) potential raise the possibility that, in addition to embryonic sources, adult valve fibroblasts may be derived from HSCs. To test this hypothesis, we used methods that allow the potential of a single HSC to be evaluated in vivo. This was achieved by isolation and clonal expansion of single lineage-negative (Lin-), c-kit(+), Sca-1(+), CD34- cells from the bone marrow of mice that ubiquitously express enhanced green fluorescent protein (EGFP) combined with transplantation of individual clonal populations derived from these candidate HSCs into a lethally irradiated congenic non-EGFP mouse. Histological analyses of valve tissue from clonally engrafted recipient mice revealed the presence of numerous EGFP+ cells within host valves. A subpopulation of these cells exhibited synthetic properties characteristic of fibroblasts, as evidenced by their expression of mRNA for procollagen 1alpha1. Further, we show by Y-chromosome-specific fluorescence in situ hybridization analysis of female-to-male transplanted mice that the EGFP+ valve cells are the result of HSC-derived cell differentiation and not the fusion of EGFP+ donor cells with host somatic cells. Together, these findings demonstrate HSC contribution to the adult valve fibroblast population.

Hematopoietic origins of fibroblasts: I. In vivo studies of fibroblasts associated with solid tumors.

OBJECTIVE: Recent studies have reported that bone marrow cells can give rise to tissue fibroblasts. However, the bone marrow cell(s) that gives rise to fibroblasts has not yet been identified. In the present study, we tested the hypothesis that tissue fibroblasts are derived from hematopoietic stem cells (HSCs) in vivo. METHODS: These studies were conducted using mice whose hematopoiesis had been reconstituted by transplantation of a clonal population of cells derived from a single enhanced green fluorescent protein (EGFP)-positive HSC in conjunction with murine tumor models. RESULTS: When tumors propagated in the transplanted mice were evaluated for the presence of EGFP(+) HSC-derived cells, two prominent populations of EGFP(+) cells were found. The first were determined to be fibroblasts within the tumor stromal capsule, a subset of which expressed type I collagen mRNA and alpha-smooth muscle actin. The second population was a perivascular cell associated with the CD31(+) tumor blood vessels. CONCLUSION: These in vivo findings establish an HSC origin of fibroblasts.

Hematopoietic origins of fibroblasts: II. In vitro studies of fibroblasts, CFU-F, and fibrocytes.

OBJECTIVE: Using transplantation of a clonal population of cells derived from a single hematopoietic stem cell (HSC) of transgenic enhanced green fluorescent protein (EGFP) mice, we have documented the hematopoietic origin of myofibroblasts, such as kidney mesangial cells and brain microglial cells. Because myofibroblasts are thought to be an activated form of fibroblasts, we tested the hypothesis that fibroblasts are derived from HSCs. MATERIALS AND METHODS: Clones of cells derived from single cells of EGFP Ly-5.2 C57Bl/6 mice were transplanted into lethally irradiated Ly-5.1 mice. Using bone marrow and peripheral blood cells from mice showing high-level multilineage hematopoietic reconstitution, we induced growth of fibroblasts in vitro. RESULTS: Culture of EGFP(+) bone marrow cells from clonally engrafted mice revealed adherent cells with morphology typical of fibroblasts. Flow cytometric analysis revealed that the majority of these cells are CD45(-) and express collagen-I and the collagen receptor, discoidin domain receptor 2 (DDR2). Reverse transcriptase polymerase chain reaction analysis of cultured cells demonstrated expression of procollagen 1-alpha1, DDR2, fibronectin, and vimentin mRNA. Fibroblast colonies consisting of EGFP(+) cells were observed in cultures of bone marrow cells from clonally engrafted mice, indicating an HSC origin of fibroblast colony-forming units. Culture of peripheral blood nucleated cells from clonally engrafted mice revealed EGFP(+) cells expressing collagen-I and DDR2, indicating that fibrocytes are also derived from HSCs. CONCLUSION: We conclude that a population of fibroblasts and their precursors are derived from HSCs.

Elevated vascular endothelial cell growth factor affects mesocardial morphogenesis and inhibits normal heart bending.

Signaling by means of vascular endothelial cell growth factor (VEGF) and its receptors (VEGFRs) is required for cardiovascular development. To examine how VEGF/VEGFR receptor signaling affects early endocardial cell behavior, embryonic quail hearts were subjected to elevated VEGF165 levels (five- to nine-somite stage). Primitive embryonic hearts microinjected with recombinant human (rh)VEGF165 exhibit several distinct malformations compared with hearts in untreated embryos: the endocardial tube is malformed with tortuous cords and folds surrounded by a diminished cardiac jelly space, and the lumens of affected hearts are conspicuously reduced. Furthermore, the embryonic heart fails to loop properly. Inhibition of bending is accompanied by an apparent failure of the dorsal mesocardium to atrophy--an event thought to be necessary for heart bending. Instead of atrophy, VEGF-treated mesocardia exhibit a marked increased in the number of resident endothelial cells. Collectively, the data suggest that the abnormally robust mesocardia in VEGF-treated hearts impede the mechanical deformation required for normal heart bending. We conclude that the excessive VEGF signaling culminates in a physical or biomechanical mechanism that acts over a wide, tissue-level, length scale to cause a severe developmental defect--failure of heart bending.

Granulocyte/macrophage origin of glomerular mesangial cells.

We previously demonstrated the ability of hematopoietic stem cells (HSCs) to generate glomerular mesangial cells by trans-planting clonal populations of cells derived from a single enhanced green fluorescent protein (EGFP)-positive HSC into lethally irradiated mice. To define more precisely the hematopoietic differentiation pathway through which mesangial cells are derived, we studied the relationship between mesangial cell expression and individual hematopoietic lineages by means of a transplantation strategy. In a series of clonal HSC transplantation experiments, we generated 3 mice engrafted predominantly by granulocytes and macrophages (GMs) and 4 mice engrafted with B-cells or with B-cells and T-cells. When the kidneys of these mice were analyzed, the mice exhibiting high GM lineage engraftment revealed much higher levels of EGFP-positive mesangial cells than those with predominantly lymphocyte engraftment. Fluorescence in situ hybridization analysis of the kidneys from a male recipient of an EGFP-positive female donor excluded cell fusion as the cause for the observed differentiation. These results support the notion that glomerular mesangial cells share their origin with GMs.